Our long-term goal is to understand the combinatorial transcriptional regulatory networks in the developing central nervous system (CNS), which play essential roles to specify a large number of distinct neuronal and glial cell types. LIM homeodomain (LIM-HD) and basic helix-loop-helix (bHLH) proteins are relatively well characterized transcription factors regulating cell type specification in the embryonic CNS. However, both the molecular mechanism by which these factors regulate gene expression and the identity of their target genes remain poorly understood. We wish to explore these important issues by specifically focusing on their role in generating spinal motoneurons (MNs). Our results demonstrate that LIM-HD factors Lhx3 and Isl1 specify two distinct neurons of the embryonic spinal cord in a combinatorial manner. Lhx3 and the LIM cofactor NLI (for nuclear LIM-interactor) form a V2-interneuron (IN)-specifying tetramer. In contrast, Lhx3 and NLI form a MN-specifying hexamer when co expressed with Isl1. During MN specification, bHLH proteins Ngn2/NeuroM are functionally synchronized with the hexamer, although the molecular mechanism underlying this novel crosstalk is not fully understood. Our recent screening effort led to the finding of genomic fragments with NLI:lsl1:Lhx3 hexamer binding sites, many of which are linked to candidate MN genes. These results led to our central hypothesis of this proposal: the hexamer directly regulates multiple MN genes in conjunction with bHLH factors to effect MN specification. We will dissect this hypothesis to uncover the molecular basis that directs MN specification in response to a combinatorial expression of transcription factors. Specifically, studies are proposed to explore the diverse functional aspects of the hexamer/bHLHs with their candidate target genes. Overall, the proposed studies should provide critical insights into the basic principles for the proper formation of the nervous system and practical information to treat a variety of neuronal injuries and diseases. Of note, LIM-HD/bHLH factors are also involved with specifying cell fates in other regions of the developing CNS (e.g., retina and forebrain) as well as non-CNS organs such as heart, pancreas and pituitary, which highlights the general importance of the proposed studies. [unreadable] [unreadable] [unreadable]